Purpose: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth.
Experimental Design: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 × 2 or 5 × 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy.
Results: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (−16%), CT-26 (−70%), and human umbilical vascular endothelial cells (HUVEC; −72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (−83%), L3.6pl (−8%), and CT-26 (−82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (−57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 × 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm3 versus 1144 mm3; CT-26: 210 mm3 versus 636 mm3; P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy.
Conclusion: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.
Radiotherapy is one of the most widely used therapeutic modalities in treatment of cancer. Local control of tumor growth is achieved by radiation-induced cell death as a result of damage to cell membranes and DNA. DNA damage can be a consequence of direct radiation effects or indirectly induced through reactive oxygen species (1, 2). These effects are not limited to tumor cells but also affect microvascular endothelial cells within the tumor stroma (3, 4). Therefore, radiosensitivity of solid tumors is not only defined by intrinsic factors such as metabolic activity and cell cycle, but also by the tumor microvascular network providing oxygen supply.
Development of a tumor microvascular network by angiogenic processes is inevitable for tumor growth and metastasis. Tumor cells produce growth factors that stimulate proliferation and migration of endothelial cells, and finally the formation of new blood vessels within the tumor tissue (5). The irregular architecture and the high permeability of tumor microvessels cause heterogeneous blood flow, high interstitial fluid pressure within the tumor, and hypoxic tumor areas. These hypoxic tumor areas are more resistant to radiotherapy despite their high vascular density (6).
Radiotherapy damages existing tumor microvessels by induction of endothelial cell apoptosis (3). These antivascular effects significantly contribute to tumor growth control by radiotherapy. In response to the endothelial damage and subsequent hypoxia, however, tumor cells increase their expression of proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (7, 8). This, in turn, leads to a rebound effect in angiongenesis after cessation of radiotherapy. Consequentially, tumors expressing high concentrations of proangiogenic growth factors are more resistant to radiotherapy (9).
Therefore, it is reasonable to believe that a combination of antiangiogenic therapy and radiotherapy may improve tumor growth control. Indeed, antiangiogenic therapies such as angiostatin, VEGF/VEGF receptor inhibitors, or epidermal growth factor receptor (EGFR) inhibitors, haven been combined with radiotherapy and have been proven to have at least additive effects on tumor growth control (8, 10–12). These additive effects have been explained by three approaches. First, as the so-called “normalizing effect” on tumor microcirculation, antiangiogenic therapy results in improved tissue perfusion and oxygen supply due to reduction of immature tumor blood vessels and reduction of oxygen-consuming endothelial cells and tumor cells (13). The second approach suggests that antiangiogenic therapy can diminish tumor repopulation after chemotherapy and radiotherapy due to interruption of the increased oxygen needs. Third, antiangiogenic therapy might suppress vasculogenesis by preventing recruitment of endothelial precursor cells in response to chemotherapy and radiotherapy (14).
Resistance of tumors to radiotherapy has been further associated with activation of distinct intracellular signaling pathways in tumor cells in response to radiation (15). In particular, radiation induces tumor cell proliferation by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway most likely through stimulation of EGFR on tumor cells (16–18). A critical downstream effector of the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR). mTOR controls translation of specific mRNA transcripts that encode proteins for cell cycle progression and cell proliferation (7, 19).
In our present study, we show that induction of mTOR inhibition 2 days before the beginning of fractionated radiotherapy resulted in improved tumor growth control in vivo. mTOR inhibitor RAD001 possesses a dose-modifying effect on radiotherapy. In vitro, proliferation of HUVEC seemed to be most sensitive to a combination of mTOR inhibition and radiotherapy, whereas tumor cells showed a cell line–specific resistance.
Hence, improved tumor growth control by combination of mTOR inhibition and radiotherapy might be based on damage to established tumor blood vessels (antivascular effect) and growth inhibition of new blood vessels in tumors (antiangiogenic effect).
Materials and Methods
Cell lines and cell culture. HUVECs were purchased from PromoCell and were maintained in polystyrene flasks (Falcon, Becton Dickinson) with growth factor–supplemented (Supplement Pack; PromoCell) endothelial-cell basal medium (PromoCell) containing 2% fetal bovine serum (FBS; Life Technologies) as detailed by the manufacturer. The murine cancer cell line CT-26 colon cancer derived from BALB/c mice was cultured in RPMI 1640 (PAN Systems) containing 10% FBS, gentamicin, sodium pyruvate, and HEPES buffer. The human pancreatic cancer cell line L3.6pl was maintained in DMEM supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, l-glutamine, vitamin solution, and penicillin-streptomycin mixture.
In vitro cell proliferation assay. HUVECs and tumor cells were cultured in 96-well microtiter plates (500-700 cells per well) in respective culture medium. mTOR inhibitor RAD001 (Everolimus; kindly provided by Novartis Institutes for Biomedical Research, Basel, Switzerland) was added to cultured cells 1 h before radiation or 24 h after radiation in escalating concentrations of 0.01, 0.1, 1, 10, or 20 ng/mL. Medium was changed every 48 h with and without addition of the mTOR inhibitor. Radiation was applied in single doses of 0.25, 0.5, 1, or 2 Gy (Mueller RT250, 225 kV, 15 mA). Cells were also treated with either the mTOR inhibitor RAD001 or radiation alone; untreated cells served as controls. Cell proliferation was assessed 5 to 6 days after radiation using the colorimetric WST-1 10% proliferation assay (Roche Diagnostics GmbH). Absorbance was measured in an ELISA reader at 450 nm 1 h after addition of WST-1.
Immunocytochemistry. For analysis of the PI3K/Akt/mTOR pathway activation in HUVECs and tumor cells, phosphorylation of the s6 ribosomal protein (s6rp) as a downstream target of mTOR and p70s6K was assessed by immunocytochemistry of cultured cells. HUVECs and tumor cells were plated on glass coverslips in completely supplemented medium; 24 h later, cells were starved in FBS and supplement-depleted diet medium for 16 h. After this conditioning period, cells were stimulated with 10% FBS; 10 ng/mL insulin-like growth factor (tumor cells); or 10 ng/mL VEGF (HUVECs) or single-dose radiation of 0.5 Gy (HUVEC), 1 Gy (CT-26), or 2 Gy (L3.6pl) with or without pretreatment with 20 ng/mL RAD001 over 1 h. Thirty minutes after stimulation, cells were fixed in methanol acidic acid. Primary rabbit anti–phospho-S6 ribosomal protein (Ser235/236) antibody (Cell Signalling Technology) was added. Detection of the primary antibody was done with a goat anti-rabbit biotinylated antibody and streptavidin (Vector). Vectashield 4′,6-diamidino-2-phenylindole (Vector) was used for staining of cell nuclei.
VEGF ELISA. For in vitro measurement of VEGF production, 4 × 104 L3.6pl human pancreatic cancer cells or CT-26 colon cancer cells were cultured in six-well culture plates for 24 h. Fresh medium only or medium supplemented with 10 ng/mL RAD001 was added, and cells were incubated for a further 1 h. Tumor cells were treated with single-dose radiation of 2, 4, or 10 Gy (Mueller RT250, 225 kV, 15 mA). Cell supernatants were harvested 72 h after radiation, and quantitative measurements of VEGF were done by ELISA (R&D Systems) according to the instructions of the manufacturer. VEGF concentration was determined spectrophotometrically at 450 nm in combination with a wavelength correction at 570 nm.
Animals. Immunodeficient male NMRI nu/nu mice (30-35 g; Harlan Winkelmann) and male BALB/c mice (20-25 g; Charles River) were used for the in vivo experiments. Immunodeficient NMRI nu/nu mice were kept under continuous laminar flow. The animals had free access to water and standard laboratory food throughout the experiments. All experimental procedures done on mice were approved by the local authorities (Regierung von Oberbayern, 55.2-1-54-2531-67/05 and 209.1-211-2531-38/04).
Orthotopic pancreatic tumor model. Human pancreatic cancer cells L3.6pl were injected orthotopically in the pancreas of immunodeficient NMRI nu/nu mice according to the method described previously (20). Briefly, after a small left abdominal incision, the spleen was exteriorized and 8 × 105 tumor cells were injected in the subcapsular region of the pancreas beneath the spleen. Tumor growth was monitored throughout the experiments by abdominal palpation and two-dimensional measurements using a caliper. Tumor volume was calculated according to the formula for ellipsoids: tumor volume = π/6 × diametershort2 × diameterlong.
Subcutaneous tumor model. Subcutaneous tumors were generated by injection of 8 × 105 tumor cells s.c. in the mid-dorsal region of the animals. CT-26 cancer cells were injected in syngenic BALB/c mice. The tumor volumes were estimated by three-dimensional measurement of subcutaneous tumors using a caliper and calculation of the volumes according to the following equation: tumor volume = a × b × c. If tumor necrosis was present, the volume of necrosis was calculated by measuring the long and short diameter of the necrotic area using the following equation: volume of necrosis = π/6 × diametershort2 × diameterlong. The volume of necrosis was subtracted from the entire tumor volume.
Experimental protocol for in vivo experiments. Fourteen to 16 days after tumor cell implantation, animals were anesthetized by inhalation of isoflurane-N2O [FiO2 0.35, 0.015 L/L isoflurane (Forene); Abbott GmbH] and fractionated radiotherapy was applied on 5 consecutive days in doses of 2 Gy (5 × 2 Gy) or 4 Gy (5 × 4 Gy; Philips RT100 250 kV, 8 mA). In the orthotopic pancreatic tumor model, animals were positioned in the right lateral position and tumors were palpated in the abdomen. The radiation beam was focused on the intra-abdominal tumor through a plexiglass tube of 1.5 cm in diameter. The maximal depth dose of radiation was delivered to the center of the pancreatic tumor, which was estimated to be located 4 mm beneath the skin in the animals.
Mice bearing subcutaneous tumors were positioned in prone position. The radiation beam was again focused through the plexiglass tube and the maximal depth dose of radiation was delivered to the center of the tumors, which was estimated to be located 3 mm beneath the skin in the animals. Radiotherapy of tumors was done with or without mTOR inhibition by RAD001. The mTOR inhibitor RAD001 was administered by daily i.p. injection of 1.5 mg/kg body weight.
RAD001 treatment was introduced 2 days before the beginning of fractionated radiotherapy, the day after the last fraction of radiotherapy, or as single therapy on day 12 (L3.6pl) or 14 (CT-26) after tumor cell implantation.
Administration of RAD001 was continued until the end of experiments. Animals were euthanized when abnormalities in behavior and ingestion occurred because of tumor burden.
Immunohistochemical determination of CD31. Frozen tissues of L3.6pl tumors were sectioned (10 μm), mounted on SuperFrost Ultra Plus slides (Menzel GmbH), and air dried for 30 min. The cryosections were fixed in cold ethanol (10 min) and washed with PBS. The sections were incubated with the primary goat–anti-mouse CD31/platelet/endothelial cell adhesion molecule 1 antibody (1:250; Santa Cruz Biotechnology) overnight at 4°C and rinsed with PBS. Sections were then incubated with the biotinylated secondary donkey–anti-goat antibody (1:200; Santa Cruz Biotechnology) for 1 h at ambient temperature. Positive reactions were visualized by incubating the slides with avidin-biotin for 1 h, followed by incubation with 3-amino-9-ethylcarbazole for an additional 30 min. The immunostained sections were counterstained with hemalaun, rinsed with distilled water, and mounted with Ultra Mount (Dako).
Statistical analysis. All results are given as mean ± SE. Data analysis was done with a statistical software package (SigmaStat for Windows; SPSS Science). One-way ANOVA test adjusted by least square difference test was used for the estimation of stochastic probability in intergroup comparison. P values <0.05 were considered to be significant.
In vitro cell proliferation. HUVECs are highly sensitive to mTOR inhibition and radiation in vitro. Even low concentrations of 0.01 ng/mL RAD001 reduced cell proliferation by 37 ± 3%; higher concentrations of the mTOR inhibitor further decreased cell proliferation by 83 ± 2% (RAD001 10 ng/mL) compared with untreated controls (Fig. 1A). Single doses of radiation decreased the proliferation of HUVECs by 17 ± 3% and 72 ± 1.5% at 0.25 and 2 Gy, respectively. The combination of RAD001 and radiation exerts additive effects on the proliferation of HUVECs in vitro. If a radiation dose of 0.25 Gy was applied to HUVECs pretreated with 0.01 ng/mL RAD001, a 57 ± 3% reduction in proliferation was observed. Interestingly, only a 29 ± 3% reduction in cell proliferation was observed when the mTOR inhibitor (0.01 ng/mL) was given after radiation (0.25 Gy). The strongest and significant reduction in proliferation of HUVECs (95 ± 1%) was achieved by combining pretreatment of 10 ng/mL RAD001 together with 2 Gy (Fig. 1A).
Human pancreatic cancer L3.6pl cells seemed to be resistant to mTOR inhibition. RAD001 caused a reduction in cell proliferation of only 8 ± 2% at a dose of 10 ng/mL. Antiproliferative effects of a single-dose radiation were limited as well: Only 16 ± 1.5% inhibition of proliferation was observed at a dose of 2 Gy (Fig. 1B). Furthermore, only a moderate increase in inhibition of L3.6pl cell proliferation was achieved by combining 10 ng/mL RAD001 and 2 Gy (24 ± 1.5%; Fig. 1B). There was no difference in inhibition of cell proliferation with respect to induction of mTOR inhibition before or after radiation.
In vitro studies on proliferation of the murine colon cancer cell line CT-26 revealed an obvious sensitivity to mTOR inhibition and radiation. Moreover, combination of both therapy modalities showed additive antiproliferative effects on CT-26 cells independent of the application of RAD001 before or after radiation. In detail, proliferation of CT-26 cells was reduced by 7 ± 2% in the presence of 0.01 ng/mL RAD001 and was further suppressed by 82 ± 1% using increasing concentrations of RAD001 (Fig. 1C). A significant reduction of cell proliferation was also achieved after application of a single-dose radiation: 0.25 Gy radiation caused an inhibition of tumor cell proliferation by 14.5 ± 1%. With higher doses of radiation, an increasing antiproliferative effect was observed, culminating in a 70 ± 1% reduction of cell proliferation at 2 Gy.
The combination of mTOR inhibition and radiation revealed additive antiproliferative effects on CT-26 colon cancer cells. A 25 ± 1% and 92 ± 0.5% reduction of cell proliferation was observed at 0.01 ng/mL RAD001 plus 0.25 Gy and 10 ng/mL RAD001 plus 2 Gy, respectively (Fig. 1C).
Immunocytochemistry. PI3K/Akt/mTOR pathway activity was studied by immunofluorescence of the downstream target phospho-s6rp in cultured cells. After starvation of cultured cells in diet medium, the PI3K/Akt/mTOR pathway seemed to be inactive as it could be shown by the absence of s6rp phosphorylation in tumor cells and HUVECs. Addition of FBS, insulin-like growth factor, or VEGF to starved cells induced phosphorylation of s6rp, confirming the activation of the PI3K/Akt/mTOR pathway. In particular, the PI3K/Akt/mTOR pathway was sufficiently activated by a single dose of radiation in tumor cells and HUVECs. Application of the mTOR inhibitor RAD001 potently blocked the phosphorylation of s6rp in response to insulin-like growth factor, VEGF, or radiation. However, even under mTOR inhibition, the presence of FBS alone resulted in some phosphorylation of s6rp in L3.6pl pancreatic cancer cells, indicating mTOR-independent pathways of s6rp phosphorylation in response to FBS in this cell line (Fig. 2).
VEGF ELISA. Radiation stimulates VEGF production by tumor cells in vitro (Fig. 3). Within 72 h after radiation, a dose-dependent increase in VEGF production by tumor cells was observed. Especially L3.6pl human pancreatic cancer cells revealed a dose-dependent increase in VEGF production that peaked in a 10-fold VEGF concentration at 10 Gy compared with controls. Radiation effects on VEGF production in L3.6pl human pancreatic cancer cells were attenuated by application of RAD001 before radiation. Radiation also stimulated VEGF production in CT-26 cells. However, the radiation-induced VEGF levels of CT-26 cells reached only ∼50% the magnitude of L3.6pl cells. Furthermore, RAD001 did not sufficiently abrogate radiation effects on VEGF production in CT-26 colon cancer cells.
In vivo tumor growth. After orthotopic implantation of human pancreatic cancer cells (L3.6pl) and s.c. implantation of murine colon cancer cells (CT-26), solid tumors developed within 5 to 7 days. In control animals, the tumor volumes after injection of L3.6pl and CT-26 cells were 1145 ± 229 mm3 (day 28) and 636 ± 136 mm3 (day 23), respectively. Treatment with mTOR inhibitor RAD001 and fractionated radiotherapy was tolerated well by the experimental animals.
A delay in tumor growth was observed after fractionated radiotherapy of 5 × 2 Gy and by daily administration of mTOR inhibitor RAD001 in all tumor entities. In the subcutaneous tumor model of CT-26 colon cancer, but not in orthotopic L3.6pl human pancreatic cancer model, monotherapy with RAD001 showed superior effects on tumor growth control compared with fractionated radiotherapy (5 × 2 Gy) alone. Combination of mTOR inhibition and fractionated radiotherapy further improved tumor growth control. In particular, if the application of RAD001 was introduced before fractionated radiotherapy of 5 × 2 Gy, tumor growth was even further reduced (L3.6pl: 352 ± 51.5 mm3 at day 39; CT-26: 200 ± 40 mm3 at day 34) compared with RAD001 given after fractionated radiotherapy (5 × 2 Gy).
High doses of fractionated radiotherapy (5 × 4 Gy) resulted in sufficient growth suppression of s.c. implanted CT-26 colon cancer (52 ± 18 mm3 at day 32) and further delay in orthotopic growth of L3.6pl pancreatic cancer (465 ± 135 mm3 at day 39). Interestingly, addition of RAD001 before 5 × 4 Gy fractionated radiotherapy had no further effect on tumor growth control in CT-26 colon cancer (116 ± 50 mm3 at day 32), but significantly suppressed growth of L3.6pl pancreatic cancer (130 ± 19 mm3 at day 39). Therefore, RAD001 exerts a dose-modifying effect on fractionated radiotherapy and improves tumor growth control at high doses of fractionated radiotherapy (Fig. 4).
Immunohistochemistry. Analysis of the tumor microvasculature by immunohistochemistry of CD31 expression in tissue sections of L3.6pl human pancreatic cancer revealed that microvascular density is reduced after treatment of tumors with the mTOR inhibitor RAD001 or fractionated radiotherapy. The combined treatment of fractionated radiotherapy of 5 × 2 Gy and the mTOR inhibitor RAD001 seemed to result in a further reduction in tumor blood vessels (Fig. 5). Upon combination of high doses of 5 × 4 Gy fractionated radiotherapy and mTOR inhibitor RAD001, the tumors were composed mainly of necrotic tissue areas.
Radiation resistance of tumors has been ascribed to several different mutations in suppressor genes, DNA damage repair mechanisms, and activation of molecular pathways (15). In particular, EGFR pathway and PI3K/Akt pathway promote radiation resistance and seem to be linked to regulation of angiogenic mechanisms at the same time. Radiation can induce activation of the EGFR family (ErbB1-3) and its receptor tyrosine kinases resulting in signal transduction through the PI3K pathway and Akt (16, 18). The subsequent phosphorylation of mTOR plays a pivotal role in regulation of translational processes via phosphorylation of the ribosomal p70s6K. Phospho-p70s6K phosphorylates the downstream s6rp, which selectively promotes translation up-regulation of mRNAs containing a 5′-terminal oligopyrimidine tract. mTOR also phosphorylates and deactivates the eukaryotic translation initiation factor (eIF)-4E binding protein (4E-BP1), which releases elongation factor 4E (elF4E; ref. 21). Furthermore, mTOR promotes translation of 5′-terminal oligopyrimidine mRNA of hypoxia-inducible factor (HIF-1). HIF-1 is well known to stimulate tumor angiogenesis and tumor growth by initiation of VEGF expression and regulation of tumor metabolism and cell cycle (22, 23).
Radiation resistance has been linked to increased HIF-1 expression either through reactive oxygen species or through activation of the PI3K/Akt/mTOR pathway (24, 25). The PI3K/Akt/mTOR pathway is negatively regulated by two tumor suppressors, the lipid phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and tuberous sclerosis complex 2 (TSC2). These tumor suppressors are frequently mutated and inactive in tumor cells, leading to constitutive activation of the PI3K pathway, Akt phosphorylation, and activation of mTOR in different types of tumors (26).
Subsequently, mTOR is an attractive target in cancer therapy because the PI3K/Akt/mTOR pathway is frequently activated in tumors promoting cell proliferation and radiation resistance. Selective mTOR inhibitors like rapamycin and its derivatives, RAD001, CCI-779, and AP23573, are already enrolled in clinical trials for cancer therapy (21, 27, 28). Tumor growth control by mTOR inhibitors has been primarily attributed to antiproliferative effects on tumor cells (29). However, the antiangiogenic properties of the mTOR inhibitor rapamycin are probably as important mechanisms against tumor growth in vivo. Application of rapamycin to tumor-bearing mice resulted in a significant reduction in microvascular density of tumors and finally in a significant tumor growth control within 2 weeks after induction of therapy. In vitro, rapamycin most effectively blocked VEGF-dependent endothelial cell proliferation compared with inhibition of tumor cell proliferation (30, 31). Furthermore, mTOR activation in ERB2-expressing breast cancer cells has been associated with a high angiogenic and metastatic potential of these tumors (31).
Our hypothesis was that addition of mTOR inhibitors to radiotherapy reduces radiation resistance and consecutively improves the antitumor efficiency compared with each therapy alone. In tumor cells, mTOR inhibition prevents the expression of proangiogenic growth factors such as VEGF, reducing the angiogenic potential of tumor cells themselves. In endothelial cells, mTOR blockade disrupts the VEGF signaling pathway, leading to inhibition of endothelial cell proliferation as well as induction of endothelial cell apoptosis. Thus, mTOR inhibition can interrupt the radiation-induced stress response of tumor cells that should protect tumor microvasculature against radiation damage (Fig. 6).
Our present study shows that combination of mTOR inhibition and fractionated radiotherapy improves tumor growth control of orthotopically implanted human pancreatic cancer (L3.6pl) and s.c. implanted mouse colon cancer (CT-26). In vitro experiments revealed that L3.6pl cells are almost resistant to radiotherapy. L3.6pl cells have been previously shown to express EGFR, which has been attributed to promote radiation resistance by activation of the PI3K/Akt/mTOR pathway (18, 20). Furthermore, mutated oncoprotein Ras contributes to radiation resistance of tumor cells by activation of downstream signaling cascades like PI3K/Akt pathway or mitogen-activated protein kinase pathway as well (32). Ras has been found to be mutated in L3.6pl cells (33). Application of the mTOR inhibitor RAD001 to tumor cells in vitro revealed a tumor cell–specific inhibition of cell proliferation. Human pancreatic cancer L3.6pl seemed to be the least sensitive to mTOR inhibition, whereas colon cancer CT-26 was characterized by a dose-dependent sensitivity to mTOR inhibition. Differences in sensitivity to mTOR inhibition of tumor cells have been previously noted and ascribed to different genetic or epigenetic mutations within the mTOR pathway, for example, regarding expression of p70s6K or elF4E, but have also been linked to the pattern of tumor suppressor genes such as p53, p27Kipl, and others (34). It has been suggested that the antiproliferative efficiency of mTOR inhibition in cancer cells depends on the aberrant activation of the PI3K/Akt pathway because of mutations in PTEN or TSC1/2 tumor suppressor genes (35). Therefore, we assume that resistance of L3.6pl to the mTOR inhibitor in vitro might be due to mutations within the PI3K/Akt/mTOR pathway downstream of PI3K. Thus, regulation of cell survival and apoptosis in L3.6pl cells might depend more on mitogen-activated protein kinase pathway than on PI3K pathway. This would explain resistance of L3.6pl to both mTOR inhibition and radiotherapy. In contrast, colon carcinoma CT-26 seemed to be sensitive to mTOR inhibition and radiotherapy in vitro. Therefore, cell survival of CT-26 might be mainly dependent on the PI3K pathway (36, 37).
In vitro, the combination of the most effective dose of radiotherapy (2 Gy) and RAD001 (10 ng/mL) led to a significant (P < 0.05) suppression of cell proliferation in all tumor cell lines under study. Interestingly, the strongest antiproliferative effect by even low concentrations of RAD001 as well as by low single dose of radiation was found in HUVECs. Moreover, the application of RAD001 before radiotherapy exerted superior antiproliferative effects on HUVECs compared with RAD001 given after radiotherapy. This effect was less pronounced in tumor cells compared with HUVECs. Thus, we believe that mTOR inhibition before radiotherapy abrogates stress response effects of radiotherapy most effectively on endothelial cells.
Radiotherapy-induced stress response in tumor cells was shown by an increased VEGF production after radiation of tumor cells in vitro. However, we evaluated a marked difference between human pancreatic cancer cells L3.6pl and colon cancer cells CT-26 regarding the range of VEGF production. L3.6pl cells released about twice the magnitude of VEGF in response to radiation compared with CT-26 cells. VEGF production was also more effectively inhibited by mTOR inhibition before radiation in L3.6pl cells compared with CT-26 cells. Hence, disruption of stress response to radiotherapy in tumor cells by mTOR inhibition might further amplify antiangiogenic effects on tumor microvascular endothelial cells.
The in vitro activation of the mTOR pathway in tumor and endothelial cells was assessed by means of s6rp phosphorylation. In general, s6rp is phosphorylated via p70s6 kinase and directly regulates translational processes upon activation of mTOR (38). Immunofluorescent staining of the phospho-s6rp in cultured tumor cells (Fig. 2) and HUVECs (data not shown) revealed activation of the PI3K/Akt/mTOR pathway after single-dose radiation. Phosphorylation of the s6rp was inhibited after application of RAD001 before radiation. Therefore, mTOR inhibition abrogates radiation-dependent activation of signaling cascades involved in translational processes and radiation resistance. However, mTOR-dependent phosphorylation of s6rp seemed to be bypassed by application of FBS to starved L3.6pl cells after RAD001 application. This might be the result of mitogen-activated protein kinase–dependent pathways of phosphorylation of ribosomal proteins, but its significance in regulation of cell proliferation is thus far not well defined (39).
Unlike the in vitro observations, the growth of orthotopically implanted L3.6pl pancreatic cancer cells was delayed by fractionated radiotherapy or application of RAD001 in vivo. A significant further delay in tumor growth occurred when application of RAD001 was initiated before fractionated radiotherapy of 5 × 2 Gy and 5 × 4 Gy. We show a dose-modifying effect of RAD001 on fractionated radiotherapy in pancreatic cancer in vivo. Combination of RAD001 and fractionated radiotherapy suppressed growth of murine CT-26 colon cancer in vivo more effectively than treatment with either therapy alone. Higher doses of radiation (5 × 4 Gy) further suppressed tumor growth in the murine tumor model. However, addition of RAD001 to higher doses of radiation showed no further therapeutic effect in the s.c. implanted colon cancer.
Tumor cell–specific sensitivity to mTOR inhibition and radiation reflecting resistance of L3.6pl human pancreatic cancer cells to both therapies was shown in vitro. Nevertheless, mTOR inhibition led to reduced tumor cell production of VEGF. Due to this fact, the significantly improved tumor growth control in vivo, in particular in orthotopic L3.6pl pancreatic tumors, cannot only be explained by antiproliferative effects on the tumor cell compartment. Vice versa, we showed a high sensitivity of HUVECs to even lower concentrations of RAD001 and lower doses of radiotherapy. These observations are in accordance with previous studies providing evidence of endothelial cell apoptosis in response to radiotherapy and antiangiogenic properties of mTOR inhibition (3, 30, 31). The immunohistochemistry of L3.6pl tumors revealed reduction of tumor blood vessels by treatment of tumors with the mTOR inhibitor. Especially, a further reduction of tumor blood vessels was seen after combined treatment of tumors with mTOR inhibitor and radiotherapy (Fig. 5).
In summary, the improved tumor growth control by combination of mTOR inhibitor RAD001 and fractionated radiotherapy in vivo might be mainly due to combined effects on growth inhibition of newly formed tumor blood vessels and damages to established tumor blood vessels by the respective single therapies. Application of mTOR inhibitor RAD001 before fractionated radiotherapy abrogates mechanisms of radiation-induced stress response and radiation resistance by blocking VEGF production in tumor cells as well as VEGF signaling in HUVECs (Fig. 6). These combined effects improve the direct anticellular effects of radiotherapy on tumor cells.
Grant support: Wilhelm Sander Stiftung (no. 2003.133.1), the Deutsche Forschungsgemeinschaft (SPP1190:64 489/3-1), and the FöFoLe Research Program (no. 436) of the University of Munich, Munich, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: P.C. Manegold and C. Paringer contributed equally to this work.